Human clinical isolates of Staphylococcus aureus, for example, strains Newman and N315, cannot grow in the absence of proline, albeit their sequenced genomes harbor genes for two redundant proline synthesis pathways. We show here that under selective pressure, S. aureus Newman generates proline-prototrophic variants at a frequency of 3 ؋ 10 ؊6 , introducing frameshift and missense mutations in ccpA or IS1811 insertions in ptsH, two regulatory genes that carry out carbon catabolite repression (CCR) in staphylococci and other Gram-positive bacteria. S. aureus Newman variants with mutations in rocF (arginase), rocD (ornithine aminotransferase), and proC (⌬ 1 -pyrroline 5-carboxylate [P5C] reductase) are unable to generate proline-prototrophic variants, whereas a variant with a mutation in ocd (ornithine cyclodeaminase) is unaffected. Transposon insertion in ccpA also restored proline prototrophy. CcpA was shown to repress transcription of rocF and rocD, encoding the first two enzymes, but not of proC, encoding the third and final enzyme in the P5C reductase pathway. CcpA bound to the upstream regions of rocF and rocD but not to that of proC. CcpA's binding to the upstream regions was greatly enhanced by phosphorylated HPr. The CCR-mediated proline auxotrophy was lifted when nonpreferred carbohydrates were used as the sole carbon source. The ccpA mutant displayed reduced staphylococcal load and replication in a murine model of staphylococcal abscess formation, indicating that carbon catabolite repression presents an important pathogenesis strategy of S. aureus infections.
Summary In bacterial two-component regulatory systems (TCSs), dephosphorylation of phosphorylated response regulators is essential for resetting the activated systems to the pre-activation state. However, in the SaeRS TCS, a major virulence TCS of Staphylococcus aureus, the mechanism for dephosphorylation of the response regulator SaeR has not been identified. Here we report that two auxiliary proteins from the sae operon, SaeP and SaeQ, form a protein complex with the sensor kinase SaeS and activate the sensor kinase’s phosphatase activity. Efficient activation of the phosphatase activity required the presence of both SaeP and SaeQ. When SaeP and SaeQ were ectopically expressed, the expression of coagulase, a sae target with low affinity for phosphorylated SaeR, was greatly reduced, while the expression of alpha-hemolysin, a sae target with high affinity for phosphorylated SaeR, was not, demonstrating a differential effect of SaePQ on sae target gene expression. When expression of SaePQ was abolished, most sae target genes were induced at an elevated level. Since the expression of SaeP and SaeQ is induced by the SaeRS TCS, these results suggest that the SaeRS TCS returns to the pre-activation state by a negative feedback mechanism.
Oxidation sensing and quorum sensing significantly affect bacterial physiology and host-pathogen interactions. However, little attention has been paid to the cross-talk between these two seemingly orthogonal signaling pathways. Here we show that the quorum-sensing agr system has a built-in oxidation-sensing mechanism through an intramolecular disulfide switch possessed by the DNA-binding domain of the response regulator AgrA. Biochemical and mass spectrometric analysis revealed that oxidation induces the intracellular disulfide bond formation between Cys-199 and Cys-228, thus leading to dissociation of AgrA from DNA. Molecular dynamics (MD) simulations suggest that the disulfide bond formation generates a steric clash responsible for the abolished DNA binding of the oxidized AgrA. Mutagenesis studies further established that Cys-199 is crucial for oxidation sensing. The oxidation-sensing role of Cys-199 is further supported by the observation that the mutant Staphylococcus aureus strain expressing AgrAC199S is more susceptible to H 2 O 2 owing to repression of the antioxidant bsaA gene under oxidative stress. Together, our results show that oxidation sensing is a component of the quorum-sensing agr signaling system, which serves as an intrinsic checkpoint to ameliorate the oxidation burden caused by intense metabolic activity and potential host immune response.
In Staphylococcus aureus, the SaeRS two-component system (TCS) encoded by the saePQRS operon controls expression of major virulence factors, such as coagulase and alpha-hemolysin. The saePQRS operon has two promoters: P1 and P3. The P1 promoter, a strong promoter, is autoinduced and can transcribe all four genes. Compared with P1, P3 shows fairly low but constitutive promoter activity, and it transcribes only saeR and saeS, the two genes encoding response regulator SaeR and sensor kinase SaeS. However, the role of each promoter in sae signaling has not been rigorously defined. In this study, we found that the genuine transcription start site (TSS) of P3 is located 78 nucleotides downstream of the previously reported TSS. Subsequently, the P3 promoter sequence was identified and validated by mutagenesis analyses. Deletion of the saePQ region including the P1 promoter did not significantly alter the expression patterns of coagulase and alpha-hemolysin, two well-known sae target genes. Due to its L18P substitution in a transmembrane domain, SaeS in strain Newman has a constitutive kinase activity. Interestingly, the mutation also rendered the protein unstable, but the protein stability was restored by SaeQ, suggesting a possible SaeQ-SaeS interaction. Ironically, the same mutation seems to increase mRNA stability. SaeR appears to be stabilized by SaeS, possibly by a proteinprotein interaction. Chromosomal mutation of P1 did not affect the expression pattern of coagulase and alpha-hemolysin. Based on these results, we conclude that transcription of saeRS from P3 is sufficient for target gene activation and that P1 is not involved in the activation.
Bacterial pathogens often employ two-component systems (TCSs), typically consisting of a sensor kinase and a response regulator, to control expression of a set of virulence genes in response to changing host environments. In Staphylococcus aureus, the SaeRS TCS is essential for in vivo survival of the bacterium. The intramembrane-sensing histidine kinase SaeS contains, along with a C-terminal kinase domain, a simple N-terminal domain composed of two transmembrane helices and a nine amino acid-long extracytoplasmic linker peptide. As a molecular switch, SaeS maintains low but significant basal kinase activity and increases its kinase activity in response to inducing signals such as human neutrophil peptide 1 (HNP1). Here we show that the linker peptide of SaeS controls SaeS’s basal kinase activity and that the amino acid sequence of the linker peptide is highly optimized for its function. Without the linker peptide, SaeS displays aberrantly elevated kinase activity even in the absence of the inducing signal, and does not respond to HNP1. Moreover, SaeS variants with alanine substitution of the linker peptide amino acids exhibit altered basal kinase activity and/or irresponsiveness to HNP1. Biochemical assays reveal that those SaeS variants have altered autokinase and phosphotransferase activities. Finally, animal experiments demonstrate that the linker peptide-mediated fine tuning of SaeS kinase activity is critical for survival of the pathogen. Our results indicate that the function of the linker peptide in SaeS is a highly evolved feature with very optimized amino acid sequences, and we propose that, in other SaeS-like intramembrane sensing histidine kinases, the extracytoplasmic linker peptides actively fine-control their kinases.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.